The har1-1 mutant of Lotus japonicus line Gifu is characterised by increased nodulation and significantly inhibited root growth in the presence of its microsymbiont Mesorhizoboium loti (for example strain NZP2235). A sexual cross between the mutant and another L. japonicus genotype Funakura (with wild-type root and nodule morphology) demonstrated Mendelian recessive segregation of both phenotypes (for root and nodule) in 242 F2 individuals. No separation of phenotypes was observed, suggesting a single mutation with pleiotropic effects. Reciprocal grafting showed that the har1-1 controlled phenotype is governed by the shoot. Using a skeletal genetic map of arbitrary molecular markers produced from a Gifu x Funakura cross, the har1-1 locus was positioned between two markers at about 7 and 13 cM distance. Single nucleotide polymorphisms (SNPs) and transgene sequences were detected by allele-specific PCR in DNA isolated from small (1 mg mass) individual seeds and half-cotyledon of the model legume Lotus japonicus, allowing fast determination of a seedling’s genomic status. This permitted a shortening of the breeding cycle for multi-trait seed lines. Fast neutron mutagenesis of Lotus japonicus wild-type genotype Gifu resulted in the first time isolation of a stable mutant (FNN5-2) unable to form nitrogen-fixing nodules in symbiosis with Mesorhizobium loti, though being infected by mycorrhizal fungi. The mutation behaves as a loss-of-function recessive, and has no other apparent phenotypic effects. Molecular characterization indicates a partial loss of the LjNFR1 LysM type receptor kinase gene. Additionally part of the LjNIN gene (encoding a putative transcription factor needed for nodulation) is also missing. Transcript levels for both genes are severely reduced. As LjNIN and LjNFR1 are in the same chromosomal region we tested whether this terminal portion is lacking. PCR analysis confirms that genes within the relevant interval (such as LjPAL1 (encoding phenylalanine ammonia lyase) and LjEIL2 (encoding an ethylene insensitive-like response regulator)) are present, suggesting that the mutational event induced by the fast neutrons was either a double hit coincidently involving two nodulation-related genes, a major genome rearrangement, or a major segmental inversion. To develop an integrated nodule developmental model based on gene interactions in autoregulation, nodulation and plant hormone response deficient lines, HE double mutants have been built using the har1-1 mutant (hypernodulation and aberrant root) and the ethylene insensitive transgenic line Etr1-1. The homozygous loss-of-function mutant har1-1 has increased nodulation and decreased root growth. Ethylene insensitivity mediated by the transgene 35S::AtETR1-1 restores the normal root growth. The HE double mutants were confirmed by triple response test and allele- or gene-specific PCR. The current results in this study indicate that a) HE double mutants shown the same nodulation pattern as har1-1 and normal root formation as Etr1-1, suggesting that nodule and root control diverge at some stage with root control being ethylene-mediated and the Har1 gene, the orthologue of GmNARK is involved in nodulation. b) Grafting demonstrated that the shoot is the source of ethylene suppression of the har1-1 induced inhibition of root growth. c) The mutated Etr1-1 gene was able to replace AVG in BAP root inhibition; d) IPT-dependent cytokinin overproduction led to aberrant root architecture in har1-1; e) Crosstalk between ethylene and cytokinin in HE double mutant by qRT-PCR.